The present invention relates in general to thermally stable and pH stable analogs of the enzyme subtilisin and to a method for generating such analogs. In particular, the present invention relates to analogs of Bacillus subtilisin having a substitution for Asn.sup.218 and to a method for generating such analogs.
The term subtilisin designates a group of extracellular alkaline serine proteases produced by various species of Bacilli. These enzymes are also called Bacillus serine proteases, Bacillus subtilisins or bacterial alkaline proteases.
The Bacillus subtilisin molecules are composed of a single polypeptide chain of either 274 residues (for subtilisin type Carlsberg produced by Bacillus licheniformis and for the subtilisin produced by Bacillus subtilis strain DY) or 275 residues (for subtilisin type BPN', produced by Bacillus amyloliquefaciens, and the aprA gene product of Bacillus subtilis). When comparing amino acid sequences of subtilisin from different strains of Bacillus the sequence of subtilisin BPN' is used as a standard. For example, based on an alignment of sequences that gives the highest degree of homology between subtilisin Carlsberg and subtilisin BPN', the serine at the active site of the former is referred to as serine 221, even though it is located at position 220 of the amino acid sequence. On the same basis, position 220 of the amino acid sequence of subtilisin Carlsberg may said to "correspond" to position 221 of subtilisin BPN'. See e.g., Nedkov et al., Hoppe-Seyler's Z. Physiol. Chem., 364, 1537-1540 (1983).
The X-ray structure of subtilisin BPN' [Wright, et al., Nature, 221, 235 (1969)] revealed that the geometry of the catalytic site of subtilisin, involving Asp.sup.32, His.sup.64 and Ser.sup.221, is almost identical to that of the active site of mammalian serine proteases (e.g., chymotrypsin) involving the residues Asp.sup.102, His.sup.57, and Ser.sup.195. However, the overall dissimilarities between Bacillus serine proteases and mammalian serine proteases indicate that these are two unrelated families of proteolytic enzymes.
In the family of Bacillus subtilisins complete amino acid sequences are available for four subtilisins: Carlsberg, [Smith, et al., J. Biol. Chem., 243, 2184-2191 (1968)]; BPN' [Markland, et al., J. Biol. Chem., 242, 5198-5211 (1967)]; the aprA gene product [Stahl, et al., J. Bacteriol., 158, 411-418 (1984)]; and DY [Nedkov, et al., supra]. Subtilisin Carlsberg and subtilisin BPN' (sometimes referred to as subtilisin Novo) differ by 84 amino acids and one additional residue in BPN' (subtilisin Carlsberg lacks an amino acid residue corresponding to residue 56 of subtilisin BPN'). Smith, et al., supra. Subtilisin DY is 274 amino acids in length and differs from subtilisin Carlsberg in 32 amino acid positions and from subtilisin BPN' by 82 amino acid replacements and one deletion (subtilisin DY lacks an amino acid residue corresponding to residue 56 of subtilisin BPN'). Nedkov, et al., supra. The amino acid sequence of the aprA gene product is 85% homologous to the amino acid sequence of subtilisin BPN'. Stahl, et al., supra. Thus, it seems that there is an extensive homology between amino acid sequences of serine proteases from different strains of Bacillus. This homology is complete in certain regions of the molecule and especially in those that play a role in the catalytic mechanism and in substrate binding. Examples of such sequence invariances are the primary and secondary substrate binding sites, Ser.sup.125 -Leu.sup.126 -Gly.sup.127 -Gly.sup.128 and Tyr.sup.104 respectively and the sequence around the reactive serine (221) Asn.sup.218 -Gly.sup.219 -Thr.sup.220 -Ser.sup.221 -Met.sup.222 -Ala.sup.223.
Subtilisin molecules exhibit some unique stability properties. They are not completely stable at any pH value although they are relatively resistant to denaturation by urea and guanidine solutions and enzymatic activity is retained for some time even in a solution of 8M urea. In solutions at a pH below 4, subtilisin rapidly and irreversibly loses its proteolytic activity. Gounaris, et al., Compt. Rend. Tray. Lab. Carlsberg, 35, 37 (1965) demonstrated that the acid deactivation of subtilisin is not due to a general charge effect and speculated that it is due to other changes in the molecule, such as protonation of histidine residues in the interior, hydrophobic parts of the molecule. In solution at pH above 5, Bacillus serine proteases gradually undergo irreversible inactivation at a rate that increases with temperature and pH. The mechanisms of this inactivation are not fully known but there is evidence indicating that autodigestion is responsible at least in part for enzyme instability at this pH range.
The use of proteases in industrial processes which require hydrolysis of proteins has been limited due to enzyme instability under operational conditions. Thus, for example, the incorporation of trypsin into laundry detergents (e.g., Bio-38, Schnyder; Switzerland) to facilitate removal of proteinaceous stains had a very limited success which was undoubtedly a result of enzyme instability under the washing conditions. It was only about 1960, after the introduction of the use of bacterial alkaline proteases which are more compatible with detergents that proteases came to be widely used in the detergent industry.
For practical reasons many industrial processes are conducted at temperatures that are above the stability range of most enzymes. Therefore, it is reasonable to assume that highly thermostable proteases not only will be advantageous to certain industries such as detergent and hide dehairing, that already require stable proteases, but may be useful in industries that use chemical means to hydrolyze proteins e.g. hydrolysis of vegetable and animal proteins for the production of soup concentrates.
It should be pointed out, however, that although thermal inactivation may be the most important mode of enzyme inactivation, factors other than heat such as extremes of pH, oxygen and denaturing agents may have a determinantal effect on limiting the use of proteases in industrial processes. It is therefore, desirable to obtain proteases that are characterized by improved stability under the operational conditions used in various industries. Such a goal may be accomplished either through searching for new more stable wild-type enzymes or through stabilization of already known existing proteases.
Even though the Bacillus-derived alkaline proteases are more compatible with detergent formulations than were the pancreatic proteases, they are still not ideal in all respects.
Over the past several years there have been major changes in detergent formulations, particularly in the replacement of phosphates with alternate builders and in the development of liquid laundry detergents to meet environmental and consumer demands. These changes create a need for changes in traditional detergent enzymes. More particularly, it has become desirable to employ proteolytic enzymes which possess greater storage stability in liquid laundry formulations as well as stability and activity at broader ranges of pH and temperature.
In one approach to producing modified subtilisins for use in detergent formulations, as disclosed in European Patent Application No. 130,756, mutations in the subtilisin of Bacillus amyloliquefaciens (B. amyloliquefaciens) at Tyr.sup.-1, Asp.sup.32, Asn.sup.155, Tyr.sup.104, Met.sup.222, Gly.sup.166, His.sup.64, Gly.sup.169, Phe.sup.189, Ser.sup.33, Ser.sup.221, Tyr.sup.217, Glu.sup.156, and/or Ala.sup.152 are identified as providing changed stability, altered conformation or as having changes in the "processing" of the enzyme. In this context mutation of Met.sup.222 to Ala, Cys (which mutant also exhibits a sharper pH optimum than wild type) or Ser assertedly results in improved oxidation stability. Substitution for Gly.sup.166 with Ala, Asp, Glu, Phe, Hys, Lys, Asn, Arg or Val appears to alter the kinetic parameters of the enzyme. However, none of the mutations are disclosed to provide analogs having greater stability at high temperatures or stability over a broader pH range than the wild type enzyme.
In another approach, it appears that the pH dependence of subtilisin may be altered, as disclosed in Thomas, et al, Nature, 318, 375-376 (1985), by changing an Asp to Ser in Asp.sup.99 -Gly.sup.100 of subtilisin BPN'. This change represents an alteration of a surface charge 14-15 Angstroms from the active site. However, the approach of Thomas, et al. does not provide an indication of improvement where no change in surface charge is made, as is the case where one uncharged residue is substituted for another.